foundations in microbiology - ltcc online · 2013-12-12 · 2 9.1 genetics and genes genetics –...

Foundations in

Microbiology Seventh Edition

Chapter 9

Microbial Genetics

Lecture PowerPoint to accompany

Talaro

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

2

9.1 Genetics and Genes

Genetics – the study of heredity

The science of genetics explores:

1. Transmission of biological traits from parent

to offspring

2. Expression and variation of those traits

3. Structure and function of genetic material

4. How this material changes

3

4

Levels of Structure and Function of

the Genome

• Genome – sum total of genetic material of a cell

(chromosomes + mitochondria/chloroplasts and/or

plasmids)

– Genome of cells – DNA

– Genome of viruses – DNA or RNA

• DNA complexed with protein constitutes the genetic

material as chromosomes

• Bacterial chromosomes are a single circular loop

• Eukaryotic chromosomes are multiple and linear

5

Figure 9.2

6

Chromosome is subdivided into genes, the fundamental unit of heredity responsible for a given trait

– Site on the chromosome that provides information for a certain cell function

– Segment of DNA that contains the necessary code to make a protein or RNA molecule

Three basic categories of genes:

1. Genes that code for proteins – structural genes

2. Genes that code for RNA

3. Genes that control gene expression – regulatory genes

7

• All types of genes constitute the genetic

makeup – genotype

• The expression of the genotype creates

observable traits – phenotype

8

Genomes Vary in Size

• Smallest virus – 4-5 genes

• E. coli – single chromosome containing

4,288 genes; 1 mm; 1,000X longer than cell

• Human cell – 46 chromosomes containing

31,000 genes; 6 feet; 180,000X longer than

cell

Figure 9.3 E. coli cell has spewed out its DNA

9

10

DNA • Two strands twisted into a double helix

• Basic unit of DNA structure is a nucleotide

• Each nucleotide consists of 3 parts:

– A 5 carbon sugar – deoxyribose

– A phosphate group

– A nitrogenous base – adenine, guanine, thymine, cytosine

• Nucleotides covalently bond to form a sugar-phosphate linkage – the backbone

– Each sugar attaches to two phosphates –

• 5′ carbon and 3′ carbon

11

DNA

• Nitrogenous bases covalently bond to the 1′ carbon of each sugar and span the center of the molecule to pair with an appropriate complementary base on the other strand

– Adenine binds to thymine with 2 hydrogen bonds

– Guanine binds to cytosine with 3 hydrogen bonds

• Antiparallel strands 3′ to 5′ and 5′ to 3′

• Each strand provides a template for the exact copying of a new strand

• Order of bases constitutes the DNA code

12

Figure 9.4

13

Significance of DNA Structure

1. Maintenance of code during reproduction

- Constancy of base pairing guarantees

that the code will be retained

2. Providing variety - order of bases

responsible for unique qualities of each

organism

14

DNA Replication • Making an exact duplicate of the DNA involves

30 different enzymes

• Begins at an origin of replication

• Helicase unwinds and unzips the DNA double helix

• An RNA primer is synthesized at the origin of replication

• DNA polymerase III adds nucleotides in a 5′ to 3′ direction

– Leading strand – synthesized continuously in 5′ to 3′ direction

– Lagging strand – synthesized 5′ to 3′ in short segments; overall direction is 3′ to 5′

15

• DNA polymerase I removes the RNA

primers and replaces them with DNA

• When replication forks meet, ligases link

the DNA fragments along the lagging strand

to complete the synthesis

• Separation of the daughter molecules is

complete

16

Figure 9.5

DNA replication is semiconservative because

each chromosome ends up with one new

strand of DNA and one old strand.

18

Figure 9.7 Completion of chromosome replication

19

20

9.2 Applications of the DNA code

• Information stored on the DNA molecule is

conveyed to RNA molecules through the

process of transcription

• The information contained in the RNA

molecule is then used to produce proteins in

the process of translation

21

22

Gene-Protein Connection

1. Each triplet of nucleotides on the RNA specifies a particular amino acid

2. A protein’s primary structure determines its shape and function

3. Proteins determine phenotype. Living things are what their proteins make them.

4. DNA is mainly a blueprint that tells the cell which kinds of proteins to make and how to make them

Figure 9.9 DNA-protein relationship

23

24

RNAs

• Single-stranded molecule made of nucleotides

– 5 carbon sugar is ribose

– 4 nitrogen bases – adenine, uracil, guanine, cytosine

– Phosphate

25

RNA • 3 types of RNA:

– Messenger RNA (mRNA) – carries DNA message

through complementary copy; message is in triplets

called codons

– Transfer RNA (tRNA) – made from DNA;

secondary structure creates loops; bottom loop

exposes a triplet of nucleotides called anticodon

which designates specificity and complements

mRNA; carries specific amino acids to ribosomes

– Ribosomal RNA (rRNA) – component of ribosomes

where protein synthesis occurs

26

27

Figure 9.10

28

Transcription:

The First Stage of Gene Expression

1. RNA polymerase binds to promoter region upstream

of the gene

2. RNA polymerase adds nucleotides complementary

to the template strand of a segment of DNA in the 5′

to 3′ direction

3. Uracil is placed as adenine’s complement

4. At termination, RNA polymerase recognizes signals

and releases the transcript 100-1,200 bases long

29

Figure 9.11

30

• All the elements needed to synthesize protein are brought together on the ribosomes

• The process occurs in five stages: initiation, elongation, termination, and protein folding and processing

Translation:

The Second Stage of Gene Expression

31

Figure 9.12

32

The Master Genetic Code

• Represented by the mRNA codons and the

amino acids they specify

• Code is universal

• Code is redundant

33

Figure 9.13

34

Figure 9.14

35

• Ribosomes assemble on the 5′ end of an mRNA transcript

• Ribosome scans the mRNA until it reaches the start codon, usually AUG

• A tRNA molecule with the complementary anticodon and methionine amino acid enters the P site of the ribosome and binds to the mRNA

Translation

36

37

Translation

• A second tRNA with the complementary anticodon fills the A site

• A peptide bond is formed

• The first tRNA is released and the ribosome slides down to the next codon

• Another tRNA fills the A site and a peptide bond is formed

• This process continues until a stop codon is encountered

38

Translation Termination

• Termination codons – UAA, UAG, and

UGA – are codons for which there is no

corresponding tRNA

• When this codon is reached, the ribosome

falls off and the last tRNA is removed from

the polypeptide

39

Figure 9.15

40

Polyribosomal complex allows for the synthesis of

many protein molecules simultaneously from the

same mRNA molecule.

41

Eukaryotic Transcription and

Translation

1. Do not occur simultaneously – transcription occurs in the nucleus and translation occurs in the cytoplasm

2. Eukaryotic start codon is AUG, but it does not use formyl-methionine

3. Eukaryotic mRNA encodes a single protein, unlike bacterial mRNA which encodes many

4. Eukaryotic DNA contains introns – intervening sequences of noncoding DNA – which have to be spliced out of the final mRNA transcript

42

Figure 9.17

43

Genetics of Animal Viruses

• Viral genome - one or more pieces of DNA

or RNA; contains only genes needed for

production of new viruses

• Requires access to host cell’s genetics and

metabolic machinery to instruct the host cell

to synthesize new viral particles

44

45

46

9.3 Regulation of Protein Synthesis

and Metabolism

• Genes are regulated to be active only when

their products are required

• In prokaryotes this regulation is coordinated

by operons, a set of genes, all of which are

regulated as a single unit

47

Operons

• 2 types of operons:

– Inducible – operon is turned ON by substrate:

catabolic operons - enzymes needed to metabolize

a nutrient are produced when needed

– Repressible – genes in a series are turned OFF by

the product synthesized; anabolic operon –

enzymes used to synthesize an amino acid stop

being produced when they are not needed

48

Lactose Operon: Inducible Operon

Made of 3 segments:

1. Regulator – gene that codes for repressor

2. Control locus – composed of promoter and

operator

3. Structural locus – made of 3 genes each coding

for an enzyme needed to catabolize lactose – b-galactosidase – hydrolyzes lactose

permease – brings lactose across cell membrane

b-galactosidase transacetylase – uncertain function

49

Lac Operon

• Normally off

– In the absence of lactose, the repressor binds with the operator locus and blocks transcription of downstream structural genes

• Lactose turns the operon on

– Binding of lactose to the repressor protein changes its shape and causes it to fall off the operator. RNA polymerase can bind to the promoter. Structural genes are transcribed.

50

Figure 9.18

51

Arginine Operon: Repressible

• Normally on and will be turned off when

the product of the pathway is no longer

required

• When excess arginine is present, it binds to

the repressor and changes it. Then the

repressor binds to the operator and blocks

arginine synthesis.

52

Figure 9.19

53

9.4 Mutations:

Changes in the Genetic Code

• A change in phenotype due to a change in genotype (nitrogen base sequence of DNA) is called a mutation

• A natural, nonmutated characteristic is known as a wild type (wild strain)

• An organism that has a mutation is a mutant strain, showing variance in morphology, nutritional characteristics, genetic control mechanisms, resistance to chemicals, etc.

54

Figure 9.20

55

Causes of Mutations

• Spontaneous mutations – random change

in the DNA due to errors in replication that

occur without known cause

• Induced mutations – result from exposure

to known mutagens, physical (primarily

radiation) or chemical agents that interact

with DNA in a disruptive manner

56

57

Categories of Mutations

• Point mutation – addition, deletion, or

substitution of a few bases

• Missense mutation – causes change in a

single amino acid

• Nonsense mutation – changes a normal

codon into a stop codon

• Silent mutation – alters a base but does not

change the amino acid

58

Categories of Mutations

• Back-mutation – when a mutated gene

reverses to its original base composition

• Frameshift mutation – when the reading

frame of the mRNA is altered

59

60

Repair of Mutations

• Since mutations can be potentially fatal, the cell has several enzymatic repair mechanisms in place to find and repair damaged DNA

– DNA polymerase – proofreads nucleotides during DNA replication

– Mismatch repair – locates and repairs mismatched nitrogen bases that were not repaired by DNA polymerase

– Light repair – for UV light damage

– Excision repair – locates and repairs incorrect sequence by removing a segment of the DNA and then adding the correct nucleotides

61

Figure 9.21

62

The Ames Test

• Any chemical capable of mutating bacterial

DNA can similarly mutate mammalian DNA

• Agricultural, industrial, and medicinal

compounds are screened using the Ames test

• Indicator organism is a mutant strain of

Salmonella typhimurium that has lost the ability

to synthesize histidine

• This mutation is highly susceptible to back-

mutation

63

Figure 9.22

64

Positive and Negative Effects of

Mutations

• Mutations leading to nonfunctional proteins are

harmful, possibly fatal

• Organisms with mutations that are beneficial in

their environment can readily adapt, survive, and

reproduce – these mutations are the basis of

change in populations

• Any change that confers an advantage during

selection pressure will be retained by the

population

65

9.5 DNA Recombination Events

Genetic recombination – occurs when an

organism acquires and expresses genes

that originated in another organism

3 means for genetic recombination in bacteria:

1. Conjugation

2. Transformation

3. Transduction

66

67

Conjugation

• Conjugation – transfer of a plasmid or chromosomal fragment from a donor cell to a recipient cell via a direct connection

– Gram-negative cell donor has a fertility plasmid (F plasmid, F′ factor) that allows the synthesis of a conjugative pilus

– Recipient cell is a related species or genus without a fertility plasmid

– Donor transfers fertility plasmid to recipient through pilus

68

Figure 9.23 (1)

69

Figure 9.23 (2)

70

Conjugation

• High-frequency recombination – donor’s

fertility plasmid has been integrated into the

bacterial chromosome

• When conjugation occurs, a portion of the

chromosome and a portion of the fertility

plasmid are transferred to the recipient

71

Figure 9.23 (3)

72

Transformation

• Transformation – chromosome fragments

from a lysed cell are accepted by a recipient

cell; the genetic code of the DNA fragment is

acquired by the recipient

• Donor and recipient cells can be unrelated

• Useful tool in recombinant DNA technology

73

Insert figure 9.23 transformation

Figure 9.24

74

Transduction

• Transduction – bacteriophage serves as a carrier of DNA from a donor cell to a recipient cell

• Two types:

– Generalized transduction – random fragments of disintegrating host DNA are picked up by the phage during assembly; any gene can be transmitted this way

– Specialized transduction – a highly specific part of the host genome is regularly incorporated into the virus

75

Figure 9.25

Generalized

transduction

76

Figure 9.26

Specialized

transduction

77

Transposons

• Special DNA segments that have the capability of moving from one location in the genome to another – “jumping genes”

• Cause rearrangement of the genetic material

• Can move from one chromosome site to another, from a chromosome to a plasmid, or from a plasmid to a chromosome

• May be beneficial or harmful

78

Figure 9.27

Transposons